TASTRIP
Research Project founded by UEFISCDI
PN-III-P1-1.1-PD-2021-0076Cell Signaling
Institute of Biochemistry
01/04/2022- 31/03/2024
Embedded Files
Functional characterization of the sweet taste receptors (Tas1R2/Tas1R3) and TRPM3 in pancreatic beta-cells.
Functional characterization of the sweet taste receptors (Tas1R2/Tas1R3) and TRPM3 in pancreatic beta-cells.
Type-2-diabetes (T2D) affects more than 400 million people worldwide and it is characterized by the inability of the body to manage glucose blood levels due to insulin resistance. G-protein coupled receptors (GPCRs) represent the largest family of membrane receptors, some of which regulate insulin release due to intracellular calcium increase through the downstream activation of voltage-gated calcium ion channels in pancreatic beta-cells. Transient receptor potential (TRP) channels constitute a class of ion channels that show permeability for sodium and calcium ions. Members of the TRP-melastatin (TRPM) family are expressed in pancreatic beta-cells and contribute to insulin release. The main objective of the proposed project is to find novel modulators of the sweet taste-G-protein coupled receptors (Tas1R2/Tas1R3) and to characterize their interaction with the heat-sensitive TRPM3 channels in pancreatic beta-cells. Both proteins contribute to glucose-dependent insulin release but their physiological functions are yet to be determined. For this purpose we will use a high-throughput screening method to identify specific modulators of the Tas1R2/Tas1R3. By monitoring intracellular calcium and glucose-dependent insulin secretion, we will be able to characterize the interactions between the metabotropic receptors and TRPM3. This approach will provide a better understanding of how these proteins relate to insulin secretion and may lead to novel therapeutic strategies.
Results
Updated on 13.06.2024
Optimization of high-throughput screening (HTS) using FlexStation 3.
HEK293T cells were transfected with human TRPM3 and the fluorescent calcium-binding protein, GCaMP6m. More than 60 FDA-approved compounds were tested for their agonistic as well as antagonistic properties.
Optimizations of multi-plasmid transfections in INS-1 cell line.
INS-1 cells were transfected with 3 plasmids: TAS1R2/TAS1R3/GCaMP6m.
Neotame and Sucralose applications increased the intracellular calcium concentration (Figure 1).
Sucralose activates a subpopulation of mouse DRG neurons in calcium imaging.
Primary neuronal cultures from mouse dorsal root ganglia were stimulated with sucralose, AITC and KCl.
6 out of 150 KCl-sensitive cells responded to the artificial sweetener (Figure 2).
Vortioxetine was tested for its potential inhibitory effect in hTRPM3.
The antidepressant vortioxetine inhibited the response to the TRPM3 agonist, CIM.
The fluorescence of HEK293T-hTRPM3/GCaMP6m was recorded in 96 wells plate. The plate was pre-incubated with Vortioxetine and naringenin for 15 minutes after which CIM was applied (Figure 3).
Deliverables
Piciu, F., Domocos, D., Chiritoiu, G., Chiritoiu-Butnaru, M., Mernea, M., Popescu, C.G., Mihai, D.P., Galateanu, B., Hudita, A., Babes, A. and Cucu, D., 2024. Transient Receptor Potential Ankyrin 1 (TRPA1) Modulation by 4-Hydroxynonenal (4-HNE) in Pancreatic Adenocarcinoma Cell Lines: Putative Roles for Therapies. Pharmaceuticals, 17(3), p.344. https://www.mdpi.com/1424-8247/17/3/344
George Opriță, Alexandru Babeș, Dan Domocoș, 2022. TAS1 receptors: An overview of their functions, expression and genetic variations. https://doi.org/10.31178/rbbs.2021.4.1.4
Poster presentation at FENS Forum 2024, Vienna 25 - 29 June. Statin effects on recombinant and natively expressed thermo-sensitive transient receptor potential ion channels.
Calcium Analyses MATLAB script. https://github.com/DanTudorDom/Calcium-Imaging.git
Figure 1
Figure 1
A. Neotame (1 mM) activated 8 INS-1 cells
B. Sucralose (5 mM) activated 24 INS-1 cells. CIM (3 uM) activated 20 out of the 24 sucralose-sensitive cells.
Figure 2
Figure 2
Sucralose ( 5 mM) activated 6 neurons from a total of 150. AITC (50 uM) activated 5 out of 6 sucralose-sensitive cells. All the sucralose-sensitive cells were sensitive to KCl (50 mM)
Figure 3
Figure 3
Stimulation of HEK293T-hTRPM3/GCaMP6m with CIM after 15 minutes pre-incubation with vortioxetine (black trace), naringenin (blue trace) and buffer (red trace).
Examples of calcium imaging data analyses using the MATLAB script (https://github.com/DanTudorDom/Calcium-Imaging.git)
Select intervals of applications
Select intervals of applications
Calculate the amplitude of each cell (delta F / F 0)
Normalize the amplitude of the second application to the first one
Plot the mean of all cells
Plot the mean of all cells
Visualize the mean and standard error of the cells
Reduce the noise
Reduce the noise
Increase the signal to noise ratio
Set thresholds in order to exclude unreliable raw data
Western Blot and ICC
2024 - Scientific Report
Activity 3.1 - Optimization of sweet taste receptors expression in heterologous system.
Procedure: INS-1 cells were transfected with human TAS1R2/TAS1R3/GCaMP6m plasmids (2 ug/ml). Transfection efficiency: 40% of the cells expressed the fluorescent protein GCaMP6m.
Results: 15 % of the KCl (50 mM) were sensitive to sucralose (5 mM), 5 % were sensitive to neotame (1 mM) and 50 % to CIM (3 uM).
Activity 3.2 - Investigation of natively expressed sweet taste receptors in primary neuronal cultures.
Procedure: Dorsal Root Ganglia neurons were dissected from mice and loaded with a synthetic calcium indicator.
Stimulations: Dissociated DRG neurons were stimulated with 5 mM sucralose for 3 minutes.
Results: 6 out of 150 KCl-sensitive cells responded to sucralose and 5 out of the sucralose-sensitive cells responded to AITC (50 uM).
Activity 3.3 - Investigation of inhibitory compounds on hTRPM3 ion channel.
Procedures: HEK293T cells were transfected with TAS1R2/TAS1R3 and the calcium indicator GCaMP6m. FlexStation 3 was used to apply the compounds and read the fluorescence.
Stimulations: Vortioxetine (30 uM), naringenin (10 uM) and buffer solutions were applied 15 min. before stimulation with the TRPM3 agonist, CIM (3 uM).
Results: The antidepressant, vortioxetine inhibited 40 % of the cell's response to CIM. Naringenin, a known inhibitor of TRPM3 was used as a positive control and inhibited 60 % of the CIM response.
2023 - Scientific Report
Activity 2.1 - Various cell lines of pancreatic origin were tested for their endogenous expression of the targeted receptors:
By using calcium imaging and specific pharmacological compounds we measured the stimulatory effect in terms intracellular calcium concentration.
Receptors: TAS1R2, TAS1R3, TRPM3, TRPA1, TRPV4, TRPM8, TRPV1.
Cell lines: PANC-1, PC-1, MIA-PaCa-2, U-87 MG, BxPC-3, INS-1, bEnd.3
Activity 2.2 - Investigation of TAS1R2/TAS1R3 endogenous expression in INS-1 pancreatic cells.
By using patch clamp and calcium imaging experiments we measured the cellular and electrical activity of INS-1 cells stimulated with the artificial sweetener, Acesulfame-k.
Acesulfame-K caused an intracellular calcium elevation that was abolished in calcium-free conditions (Fig. 2).
In voltage-clamp, whole cell configuration, Acesulfame-k produced inward currents that ware diminished by the CaCl channels antagonists, MONNA.
Activity 2.3 - High-throughput screening optimization for identifying novel modulator for the TRPM3 ion channel.
HEK293t cells were transfected with human TRPM3 and GCaMP6m in 96 well plates. Pharmacological compounds from different libraries were applied on the cells after 20 seconds of background reading. The fluorescence determined by intracellular calcium was measured using Flex Station 3.
Until the 1st of December 2023, more than 50 pharmacological compounds were tested for their ability to initiate calcium influx in HEK293t/hTRPM3 cells.
For antagonistic properties of putative compounds, selective TRPM3 agonists were added in each well and the fluorescence was again measured on the whole plate.
2022 - Scientific Report
Activity 1.1 - Optimization of sweet taste receptors expression in heterologous systems.
The sweet taste receptors plasmids where purchased from Addgene together with the empty vector:
pcDNA3.1(+)-HA-Flag-natT1R2;
pcDNA3.1(+)-HLA-Flag-natT1R3;
pcDNA3.1-HA.
The plasmids were transfected in HEK293t cells using jetPei transfection reagent in a proportion of 1:1.
For identifying the expression level of the synthetized protein we used Western Blot and Immunohistochemistry (ICC) with anti-FLAG antibody (Fig. 1)
Activity 1.2: - Functional assay optimization for possible TAS1R2/TAS1R3 modulators.
To determine the functional activity of TAS1R2/TAS1R3 various cell lines (HEK293t, COS-7, CHO) were transfected with the receptors using jetPei and Lipofectamine 3000 reagents.
Both calcium and cAMP probes were used to measure the intracellular activity of the transfected cells while stimulated with artificial sweeteners (sucralose, acesulfame-k, neotame)
Project Team
Project Team
Project Leader: Dan Domocos, PhD
Project Mentor: Stefana Petrescu, PhD
Volunteers:
Researcher: George Oprita, PhD student
Researcher: Teodora Stratulat, PhD student
Project Chapters
Project Chapters
I
Identifying novel specific agonists for Tas1R2 and Tas1R3 receptors.
Task 1.1 Heterologous expression of Tas1R2/Tas1R3 receptors.
Task 1.2 Optimization of HTS strategies.
II
Validation of the lead compounds on endogenously expressed Tas1R2 and Tas1R3.
Task 2.1 Validation of the agonistic properties of the lead compounds identified in Objective 1 on natively expressed Tas1R2/Tas1R3.
Task 2.2 Further corroboration of the lead compounds on primary cell cultures.
Task 2.3 Effects of Tas1R2/Tas1R3 activation on insulin release.
III
Characterization of the interaction between Tas1R2/Tas1R3 and TRPM3.
Task 3.1 Expression analyses of the co-expression of TRPM3 channels and Tas1R2/Tas1R3 in β-cell lines.
Task 3.2 Characterisation of the interaction between Tas1R2/Tas1R3 and TRPM3.
Task 3.3 Interfering with the functional expression of Tas1R2/Tas1R3 and TRPM3 in pancreatic β-cells.
Project Deliverables
Project Deliverables
Review Article: TAS1 receptors. An overview of their functions, expression and genetic variations. Opriță George, Babeș Alexandru, Domocoș Dan; Rev. Biol. Biomed. Sci. 2021 4 (1) 59-67;
DOI: 10.31178/rbbs.2021.4.1.4
Domocos Dan Tudor, PhD
Web of Science ResearcherID: GRR-9591-2022
ORCiD: 0000-0003-2574-253X
Brain Map ID: U-1700-032J-5857
Researchgate: https://www.researchgate.net/profile/Dan-Domocos-2
Publications:
Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla in mice Published: Aug 2022 in eLife
DOI: 10.7554/ELIFE.69626
The Association between TRP Channels Expression and Clinicopathological Characteristics of Patients with Pancreatic Adenocarcinoma Published: Aug 2022 in International Journal of Molecular Sciences DOI: 10.3390/IJMS23169045
Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells (vol 11, 2018, 2021) Published: Apr 2021 in Scientific Reports DOI: 10.1038/S41598-021-88169-9
Role of 5-HT1A and 5-HT3 receptors in serotonergic activation of sensory neurons in relation to itch and pain behavior in the rat Published: Oct 2020 in Journal of Neuroscience Research DOI: 10.1002/JNR.24633
Transient receptor potential ankyrin 1 (TRPA1) positively regulates imiquimod-induced, psoriasiform dermal inflammation in mice Published: Jul 2019 in Journal of Cellular and Molecular Medicine DOI: 10.1111/JCMM.14392
Signs of chronic itch in the mouse imiquimod model of psoriasiform dermatitis: sex differences and roles of TRPV1 and TRPA1 Published: Jul 2019 in Itch
DOI: 10.1097/ITX.0000000000000025
TRPV1 mediates inflammation and hyperplasia in imiquimod (IMQ)-induced psoriasiform dermatitis (PsD) in mice Published: Dec 2018 in Journal of Dermatological Science DOI: 10.1016/J.JDERMSCI.2018.11.009
Career Summary:
My PhD thesis concerned the effects of serotonin (5-HT) on the peripheral nervous system with implications in itch and pain. In my last five years of scientific research I studied the molecular mechanisms of how touch and pain signals are received and transmitted by the nervous system. My work in the laboratory of Professor Alexandru Babes was mainly focused on thermosensitive ion channels like TRPA1, TRPV1 or TRPM8 expressed in dorsal root and trigeminal ganglia neurons. The investigations that I conducted for my PhD thesis used calcium mircofluorimetry, patch-clamp, cell transfection techniques and behavioral models of itch and pain. By combining these techniques with selective agonists and antagonists for 5-HT receptors I was able to identify the receptor subtypes expressed in rat DRG neurons. These receptor subtypes are responsible for itch signal transduction in the peripheral nervous system.
Projects
PN-III-P1-1.1-PD-2021-0076 2022 - 2024
Functional characterization of the sweet taste receptors (tas1r2/tas1r3) and trpm3 in pancreatic beta-cells.
Coordinating institution:
INSTITUTUL DE BIOCHIMIE (RO)
PN-III-P4-ID-PCE-2020-2411 2021 - 2023
Identification and characterization of the cart-neuropeptide receptor
Coordinating institution:
INSTITUTUL DE BIOCHIMIE (RO)
Project website: https://www.cartp.net/
PN-III-P1-1.2-PCCDI-2017-0797 2018 - 2021
Pathogenic mechanisms and personalized treatment in pancreatic cancer using multi-omics technologies
Coordinating institution:
INSTITUTUL CLINIC FUNDENI (RO)
Project website: https://cemt.ro/proiect/pancngs/ https://cemt.ro/en/proiect/pancngs-2
PN-III-P4-ID-PCE-2016-0475 2017 - 2019
Coordinating institution:
UNIVERSITATEA BUCURESTI (RO)
Molecular targets responsible for the pain associated with neurovisceral porphyrias
Research
DRG, TG and cortical primary neuronal cultures.
Calcium and voltage imaging.
Patch-Clamp.
Intracellular signaling.
Behavior
The signaling pathway of the TAS1R heterodimer activation. When a ligand binds to the TAS1R2-TAS1R3 dimer, the G-protein β3γ13-subunits subunits induce Ca2+ release from the ER via the PLCβ2 and IP3 pathway. The rise in intracellular calcium induces TRPM4 and TRPM5 activation which leads to sodium influx and ATP release via CALHM1 and CALHM3. Further sodium influx is mediated by VDSCs. The alpha-gustducin subunit induces an increase in cAMP and the activation of PKA, which phosphorylates and thus inhibits VDKCs.
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